The objective of this proposal is to experimentally determine the pathways affected by essential protein- coding genes in E. coli that have not yet been assigned a function. Of the ~4,300 E. coli open reading frames, roughly a third have not had their encoded protein functions experimentally verified. During this project, we plan to make significant advances in our understanding of the functions of five of these enigmatic genes that represent challenging and potentially rewarding subjects. A project is presented that will allow, for the first time, the characterization of cell physiology in the absence of conserved, essential proteins that have otherwise been experimentally intractable. Additionally, we intend to reveal the biochemical pathways influenced by these proteins by monitoring transcriptional responses to their depletion. A portion of the proposed work will be performed by undergraduate students participating in an applied research program. Once completed, the proposed research will have shed new light on the function of several essential, uncharacterized genes that are important for life and will provide a solid experimental framework for assigning gene function that extends significantly beyond computational prediction. The project goals are to (1), determine the viability of cells depleted of the proteins encoded by five essential, conserved genes. Each of these genes is conserved throughout bacteria and have many homologs in archaea and eukarya; and (2), determine the biochemical pathways most affected by the depletion of the proteins encoded by those same five genes. The rapid depletion of each essential protein will manifest a unique biochemical limitation that will be reflected in a rapid change in physiology related to the most affected pathways. This reflex will be used to assign a signature response to the loss of the essential protein and to guide the preliminary assignment of its function. PUBLIC HEALTH RELEVANCE: Many genes are conserved in all organisms because they are necessary for the fundamental processes that make life possible. Unfortunately, we still do not know the function of many of these genes despite their importance. This project will reveal the cellular pathways that are affected by a sub-set of conserved, essential genes in a bacterial model system so that we can finally gain insight into their biological roles.